The purpose of this R21 application is to explore the feasibility of using bacteriophage resistance as a feature of a live, attenuated, oral Listeria monocytogenes vaccine delivery platform. We are studying a phage resistant mutant in which a Tn917 insertion interrupts a gene whose product is required for the production of the phage receptor. This lesion also results in profound attenuation but retention of the immunostimulatory characteristics for which L. monocytogenes is well known. We intend to use the funding requested to conduct proof-of-principle experiments that will determine the usefulness of a derivative of this strain in expressing a heterologous antigen. The antigen we have chosen to express is the early secretory antigenic target 6 (ESAT-6) a secreted protein produced by Mycobacterium tuberculosis whose properties as a protective immunogen are well established. Several properties of the phage resistant mutant revealed its potential value as a vaccine platform: First, the mutant made minute plaques on cultured mouse enterocytes, indicating that it was not attenuated to the point where it would be nonimmunogenic. Second, the mutant prospered in the mouse intestinal tract following oral administration, but could not be detected in the liver and spleen--features that speak to the applicability, practicality, and safety of the mutant. Third, the parent strain we have employed is highly infective for mice via the oral route, making it a good model for studies of oral (as opposed to parenteral) listerial immunization. The strain is additionally advantageous because it is serologically similar to the most prevalent human epidemic isolates. Consequently, the development of a vaccine platform using this strain will have added value in protecting susceptible populations against infection with L. monocytogenes, a potential bioterror agent. In our protection studies, mice challenged at 22 and 52 days post vaccination had a 10,000-fold stimulation in the adaptive immune response--measured as splenic colony forming units recovered from vaccinated versus sham vaccinated mice 5 days post oral challenge with the virulent parental (phage sensitive) strain. In this application, we propose experiments to construct a L. monocytogenes phage resistant expression platform, and conduct tests of its safety and efficacy. Specifically, we propose to: (1) Construct a gene fusion strain expressing the Mycobacterium tuberculosis antigen ESAT-6 and document the level and type of immunity engendered to ESAT-6 following oral vaccination in mice. (2) Assess the level of protective immunity conferred against an aerosol M. tuberculosis challenge in mice orally vaccinated with the ESAT-6-expressing strain. A demonstration of protective immunity to M. tuberculosis by the platform would provide justification for testing antigens from other infectious agents (e.g., viral), and tumor antigens--where the platform could be applied as a therapeutic adjunct to combat certain forms of cancer--to the benefit of human health in the US. PUBLIC HEALTH RELEVANCE: The purpose of this R21 application is to explore the feasibility of using bacteriophage resistance as a feature of a live, oral, Listeria monocytogenes vaccine delivery platform. We propose means to construct the expression platform, and conduct tests of its safety and efficacy in preventing tuberculosis in mice. A demonstration of protective immunity by the platform would provide justification for testing antigens from other infectious agents (e.g., viral) and exploring the platform's applicability as a therapeutic adjunct to combat certain forms of cancer-- all to the benefit of human health in the US.